Most eukaryotic pre-mRNAs contain non-coding sequences (introns) that must be removed in order to accurately place the coding sequences (exons) in the correct reading frame. This critical regulatory event, termed pre-mRNA splicing, is fundamental in development and cancer, and occurs in a multi-component macromolecular machine, the spliceosome. Although the mechanism of pre-mRNA splicing has been extensively studied, the structure and regulation of this process is still not well understood. We have studied the mammalian splicing complex in its intact form, isolated from nuclei of living cells. The complexes we have isolated from cell nuclei are much larger than the splicing complexes assembled in vitro, and are thus termed supraspliceosomes. We have recently shown that the supraspliceosome is composed of four active native spliceosomes, each resembling the in vitro assembled spliceosome, which are connected via the pre-mRNA. Health Relevance: Defects in alternative splicing were correlated with human pathologies and malignancy. It is therefore anticipated that better understanding of the mechanism of pre-mRNA splicing should lead to better understanding of development and cancer. The long term objective of this project is to understand the regulation of splicing and alternative splicing, thus recognizing how defects in these important processes affect human diseases. Hypothesis: The isolated supraspliceosomes represent the steady-state population of nuclear pre-mRNAs that were isolated at different stages of the splicing reaction. It is thus assumed that developing methods for the preparation and isolation of homogeneous supraspliceosomes with respect to their transcript and splicing stage should allow us to get a higher resolution of the structure, a better knowledge of the components, localization and interactions within the supraspliceosome, and thus a better understanding of the working of the RNA splicing machine. Specific Aims: We propose to conduct in-depth structural and functional analyses of the native spliceosome and the supraspliceosome, including higher resolution structural analysis by the cryo-EM single particle techniques. We will perform analyses of native spliceosomes and supraspliceosomes assembled on specific transcripts, including alternatively spliced transcripts, and trapped in specific functional states (SA#1). We will also perform proteomic analyses by mass spectrometry of these complexes (SA#2). This approach might reveal differences in the composition of spliceosomes that were arrested at specific splicing stages or associated with different pre-mRNAs. Localization of spliceosomal components within supraspliceosomes and native spliceosomes derived from them will be performed using nucleic acids and antibodies tagged with gold-nanoclusters (SA#3), followed by cryo-EM structural analyses. The structural analyses will also include supraspliceosomes reconstituted with gold-tagged pre-mRNA. These experiments should assist in identifying the pre-mRNA pathway within the assembled complex and enable the localization of key spliceosomal components. PUBLIC HEALTH RELEVANCE: Defects in alternative splicing were correlated with human pathologies and malignancy. It is therefore anticipated that better understanding of the mechanism of pre-mRNA splicing should lead to better understanding of development and cancer. The long term objective of this project is to understand the regulation of splicing and alternative splicing, thus recognizing how defects in these important processes affect human diseases.